The long-term objectives of the candidate (Dr. Joshua B. Benoit) are to become an authority on the physiology of insect disease vectors and increase public awareness of the role of insects on public health. The goal of this fellowship is to develop the candidate's ability to assess the biochemistry, molecular biology and physiology of vector reproduction. The training plan provides opportunities to conduct extensive studies at the interface between molecular biology and reproductive physiology. Tsetse flies are the sole vector of African sleeping sickness. Vaccine-based methods have not been successful for control of trypanosomiasis. A unique aspect of this disease vector is its K-strategy of reproduction. Mothers provide nutrients, directly to their intrauterine progeny via a milk gland and only give birth to 8-10 offspring in their lifetime. Interference with tsetse reproduction can supplement trapping and other control methods used to reduce tsetse population numbers and decrease disease transmission. Currently, there is little information on tsetse reproduction, particularly how nutrients are mobilized to the larva. Since this aspect is unique in tsetse, control techniques blocking maternal nutrients could be tsetse specific. The objectives of this study are to 1) characterize the metabolism and transfer of lipids from the mother to her offspring, and 2) decipher how this process is influenced by the presence of symbiotic bacteria. Characterization of lipid metabolism and mobilization will focus on the adipokinetic hormone (AKH; this portion has been completed) and Brummer (Bmm) lipase systems, as well as lipid transport by lipophorin (Lp) during the reproductive cycle. Gene expression and protein synthesis will be assessed spatially and temporally during larval intrauterine development. Function of these proteins will be assessed utilizing RNA interference to knockdown their expression. Role of symbiotic bacteria in lipid metabolism and movement during reproduction will be assessed by analyzing molecular and physiological differences between wild type and antibiotic-treated aposymbiotic flies (flies lacking symbionts). Aposymbiotic flies are infertile and abort undeveloped larva early in their developmental cycle. The mechanism(s) underlying this phenotype are unknown, but malnourishment due to milk secretions deficient in essential nutrients is the current hypothesis. A possible deficiency is in the lipid content of the milk. Differential lipid composition between wild type and aposymbiotic flies will be assessed by obtaining milk and comparing the quantitative and qualitative differences in milk lipids. Fertility can be restored in aposymbiotic flies by supplementation of blood meals with yeast extract. Lipid compositions of aposymbiotic flies with and without yeast extract-supplemented blood will also be performed. To determine genetic effects of dietary supplementation, wild-type and aposymbiotic flies will be compared by Illumina sequencing of whole body transcriptome. This project will provide avenues to develop proficiency in multiple molecular and physiological techniques that will provide the groundwork for the candidate to become an authority on the molecular physiology of disease vectors.